Development of a Microgram Scale Video-Microscopic Method to Investigate Dissolution Behavior of Poorly Water-Soluble Drugs.

Poor aqueous solubility is a common characteristic of new drug candidates, which leads to low or inconsistent oral bioavailability. This has sparked an interest in material efficient testing of solubility and dissolution rate. The aim was to develop a microgram scale video-microscopic method to screen the dissolution rates of poorly water-soluble drugs. This method was applied to six drugs (carvedilol, diazepam, dipyridamole, felodipine, fenofibrate, and indomethacin) in fasted state simulated intestinal fluid (FaSSIF), of indomethacin in buffer with varying pH, and of diazepam and dipyridamole in customized media. An additional aim was to track phase transformations for carbamazepine in FaSSIF. The dissolution rates and particle behavior of the drugs were investigated by tracking particle surface area over time using optical video-microscopy. Applying miniaturized UV spectroscopic dissolution resulted in a similar grouping of dissolution rates and pH effects, as for the video-microscopic setup. Using customized media showed that lysophospholipid enhanced the dissolution rate of diazepam and dipyridamole. The video-microscopic setup allowed for the nucleation of transparent particles on dissolving carbamazepine particles to be tracked over time. The developed setup offers a material efficient screening approach to group drugs according to dissolution rate, where the use of optical microscopy helps to achieve a high sample throughput.

Visualizing the Journey of Fenofibrate through the Rat Gastrointestinal Tract by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Mapping the spatial distribution of a drug throughout the gastrointestinal tract (GIT) after oral ingestion can provide novel insights into the interaction between the drug, the oral drug delivery system, and the GIT. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a molecular imaging technique that can analyze molecules in the cryosections of tissues, determining their localization with a spatial resolution of 10-100 µm. The overall aim of this study was to use MALDI-MSI to visualize the distribution and spatial location of a model prodrug (fenofibrate) through the rat GIT. Furthermore, the distribution and spatial colocalization of taurocholate and phospholipids in the rat GIT in relation to fenofibrate were investigated. Rats were given a fenofibrate suspension of 10 mg/mL by oral gavage. Blood samples were drawn, and the rats were euthanized at three different time points. The GIT was collected and frozen, and MALDI-MSI was applied on cross sections of the stomach and intestine. Fenofibrate was detected by MALDI-MSI throughout the GIT, which also revealed that fenofibrate was hydrolyzed to the active drug fenofibric acid already in the stomach. Furthermore, the presence of lyso-phosphatidylcholine (lyso-PC) and taurocholate was confirmed in the lumen of the small intestine. MALDI-MSI was shown to be a useful qualitative tool for localizing parent prodrugs and active drugs, with a possibility for gaining insight into not only the location for activation but also the role of endogenous molecules in the process.

Development of a Video-Microscopic Tool To Evaluate the Precipitation Kinetics of Poorly Water Soluble Drugs: A Case Study with Tadalafil and HPMC.

Many drug candidates today have a low aqueous solubility and, hence, may show a low oral bioavailability, presenting a major formulation and drug delivery challenge. One way to increase the bioavailability of these drugs is to use a supersaturating drug delivery strategy. The aim of this study was to develop a video-microscopic method, to evaluate the effect of a precipitation inhibitor on supersaturated solutions of the poorly soluble drug tadalafil, using a novel video-microscopic small scale setup. Based on preliminary studies, a degree of supersaturation of 29 was chosen for the supersaturation studies with tadalafil in FaSSIF. Different amounts of hydroxypropyl methyl cellulose (HPMC) were predissolved in FaSSIF to give four different concentrations, and the supersaturated system was then created using a solvent shift method. Precipitation of tadalafil from the supersaturated solutions was monitored by video-microscopy as a function of time. Single-particle analysis was possible using commercially available software; however, to investigate the entire population of precipitating particles (i.e., their number and area covered in the field of view), an image analysis algorithm was developed (multiparticle analysis). The induction time for precipitation of tadalafil in FaSSIF was significantly prolonged by adding 0.01% (w/v) HPMC to FaSSIF, and the maximum inhibition was reached at 0.1% (w/v) HPMC, after which additional HPMC did not further increase the induction time. The single-particle and multiparticle analyses yielded the same ranking of the HPMC concentrations, regarding the inhibitory effect on precipitation. The developed small scale method to assess the effect of precipitation inhibitors can speed up the process of choosing the right precipitation inhibitor and the concentration to be used.

Investigation of the Intra- and Interlaboratory Reproducibility of a Small Scale Standardized Supersaturation and Precipitation Method.

The high number of poorly water-soluble compounds in drug development has increased the need for enabling formulations to improve oral bioavailability. One frequently applied approach is to induce supersaturation at the absorptive site, e.g., the small intestine, increasing the amount of dissolved compound available for absorption. However, due to the stochastic nature of nucleation, supersaturating drug delivery systems may lead to inter- and intrapersonal variability. The ability to define a feasible range with respect to the supersaturation level is a crucial factor for a successful formulation. Therefore, an in vitro method is needed, from where the ability of a compound to supersaturate can be defined in a reproducible way. Hence, this study investigates the reproducibility of an in vitro small scale standardized supersaturation and precipitation method (SSPM). First an intralaboratory reproducibility study of felodipine was conducted, after which seven partners contributed with data for three model compounds; aprepitant, felodipine, and fenofibrate, to determine the interlaboratory reproducibility of the SSPM. The first part of the SSPM determines the apparent degrees of supersaturation (aDS) to investigate for each compound. Each partner independently determined the maximum possible aDS and induced 100, 87.5, 75, and 50% of their determined maximum possible aDS in the SSPM. The concentration-time profile of the supersaturation and following precipitation was obtained in order to determine the induction time (tind) for detectable precipitation. The data showed that the absolute values of tind and aDS were not directly comparable between partners, however, upon linearization of the data a reproducible rank ordering of the three model compounds was obtained based on the ß-value, which was defined as the slope of the ln(tind) versus ln(aDS)-2 plot. Linear regression of this plot showed that aprepitant had the highest ß-value, 15.1, while felodipine and fenofibrate had comparable ß-values, 4.0 and 4.3, respectively. Of the five partners contributing with full data sets, 80% could obtain the same rank order for the three model compounds using the SSPM (aprepitant > felodipine ≈ fenofibrate). The α-value is dependent on the experimental setup and can be used as a parameter to evaluate the uniformity of the data set. This study indicated that the SSPM was able to obtain the same rank order of the ß-value between partners and, thus, that the SSPM may be used to classify compounds depending on their supersaturation propensity.

Exploring the Impact of Intestinal Fluid Components on the Solubility and Supersaturation of Danazol.

Eleven simulated intestinal fluids (SIF) were designed using a Design of Experiment (DoE) approach. The DoE SIF covered a range of compositions of fasted state human intestinal fluid (FaHIF) with regard to pH, bile salt (BS), and phospholipid (PL). Using the model compound danazol, the apparent crystalline solubility (aCS) and apparent amorphous solubility (aAS), as well as the supersaturation propensity was determined in the DoE SIF media. The aCS of danazol was dependent on the composition of the SIF, with PL as the main factor, and a small effect from BS and an interaction between BS and PL. From the DoE solubility data a model was derived, which could predict aCS in commercially available SIF (FaSSIF-V1 and -V2) and in a range of FaHIF. The aAS of danazol was differently affected by the SIF composition than the aCS; PL was again the main factor influencing the aAS, but interactions between BS and pH, as well as pH and PL were also important. The supersaturation propensities of danazol in the DoE SIF media were affected by the same factors as the aCS. Hence, the supersaturation behaviour and aCS of danazol, were found to be closely related.

Predicting in vivo performance of fenofibrate amorphous solid dispersions using in vitro non-sink dissolution and dissolution permeation setup.

Amorphous solid dispersion (ASD) is emerging as a useful formulation strategy to increase the bioavailability of active pharmaceutical ingredients with poor solubility. In vitro dissolution testing under non-sink conditions has often been used to evaluate the ability of ASDs to generate and maintain supersaturation to predict the in vivo performance. However, such a single compartment dissolution setup can fail to predict the oral bioavailability, due to an interdependence between precipitation and permeation. Hence, the use of two compartment dissolution-permeation setups is emerging. In this study, three ASDs containing fenofibrate as model drug substance were developed using Soluplus®, and Hypromellose Acetate Succinate in two different grades (high and low), respectively. The aim was to compare the use of a small-scale in vitro non-sink dissolution setup and a small-scale in vitro dissolution-permeation setup to predict the in vivo oral exposure of the ASDs in rats. The maximum concentration (Cmax) and area under curve (AUC) obtained in the in vitro studies were used to predict the in vivo rank order of the formulations. The results showed that the two in vitro studies resulted in the same rank order based on both Cmax and AUC. Interestingly, Cmax resulted in a better in vitro/in vivo correlation than the in vitro AUC, and based on the in vitro Cmax, the in vivo rank order was predicted.

Comparison of induction methods for supersaturation: Amorphous dissolution versus solvent shift.

Simple solvent shift is often used to induce supersaturation and investigate precipitation kinetics in early drug development as a substitute for amorphous dissolution. This study develops and compares a small-scale non-sink amorphous dissolution method to a solvent shift method as induction methods for supersaturation of the model drugs albendazole, felodipine and tadalafil with respect to the maximum dissolved drug concentration, and the solid form of the precipitate. The study also investigates the effect of pre-dispersed precipitation inhibitors (hydroxypropyl methyl cellulose (HPMC) or polyvinylpyrrolidone (PVP)) on tadalafil supersaturation induced by both amorphous dissolution and solvent shift with respect to maximum dissolved drug concentration, precipitation rate and solid form of the precipitate. The maximum drug concentrations achieved through solvent shift were 15.9, 208 and 108 µg/mL for albendazole, felodipine and tadalafil, respectively. Pre-dispersing 0.1% (w/v) HPMC or PVP, increased the maximum concentration by solvent shift of tadalafil to 180 µg/mL, for both polymers. Dissolution of up to 90 mg albendazole, 120 mg felodipine and 8.9 mg tadalafil could yield a maximum dissolved drug concentration of 76.1%, 87.9% and 102.5%, respectively, of the corresponding solvent shift maximum concentration. The maximum concentration achieved through amorphous dissolution of tadalafil with HPMC or PVP present in the dissolution medium was 87.1% and 88.7%, respectively of the solvent shift maximum concentration. Dissolution of 2 mg amorphous tadalafil with and without pre-dispersed polymer gave the same rank order of onset of precipitation as for the solvent shift method. The solid form of precipitate was the same for albendazole, felodipine, tadalafil and tadalafil with PVP for both methods. For tadalafil with HPMC, the precipitate was amorphous following solvent shift, but crystalline after amorphous dissolution. Overall, this study shows that the maximum concentration achievable through amorphous dissolution can be estimated when performing solvent shift and the precipitation inhibition of excipients assessed via solvent shift can be used to predict the effect on precipitation using amorphous dissolution.

Comparison of induction methods for supersaturation: pH shift versus solvent shift.

In order to investigate the supersaturation propensity of drugs in a simple and small-scale setup, induction of supersaturation is often performed via solvent shift. For weak bases, a potentially more biorelevant induction method would be a pH shift, as it is often hypothesized that the induction method will impact the supersaturation and precipitation. However, this has not been investigated systematically in a pharmaceutical context. This study investigates the impact of the induction method of supersaturation for nine basic drugs, A0619, A2853, albendazole, cinnarizine, dipyridamole, intraconazole, JNJ39393406, ketoconazole, and posaconazole, on four parameters: highest apparent degree of supersaturation, induction time, precipitation rate and solid form of the precipitate, using a novel standardized small-scale supersaturation and precipitation method. For eight of the nine drugs, the same highest apparent degree of supersaturation was obtained with both induction methods. For the induction time, precipitation rate and the solid form of the precipitate, no systematic differences between the induction methods were detected, however individual drugs did show differences. The study shows that, if a drug is sufficiently soluble at low pH, the solvent shift and pH shift induction methods of supersaturation yield comparable results.

Effect of supersaturation on absorption of indomethacin and tadalafil in a single pass intestinal perfusion rat model, in the absence and presence of a precipitation inhibitor.

The effect of the degree of supersaturation (DS) on absorption of the model drugs indomethacin and tadalafil was elucidated in a single-pass intestinal perfusion (SPIP) model in rats. In addition, the performance of the precipitation inhibitor (PI) hydroxypropylmethylcellulose (HPMC) was evaluated when added at a concentration of 0.1% (w/v) to fasted state simulated intestinal fluid (FaSSIF and FaSSIFHPMC) used as perfusion medium. A supersaturated state was created by a solvent shift method where indomethacin or tadalafil dissolved in dimethyl sulfoxide (DMSO) were administered to a segment of the small intestine, which subsequently was perfused with FaSSIF or FaSSIFHPMC. The perfusate was collected for 60 min, and for one group of rats dosed with 30 mg tadalafil, for 120 min. Blood samples were drawn every 15 min. The solubility of indomethacin and tadalafil in the perfusate was determined. The DS of each drug in the perfusate was calculated by dividing the concentration in the perfusate at selected time points with the solubility. The DS was above one for all timepoints for both drugs, thus showing supersaturation during the time of perfusion. For indomethacin, no improvement of the DS was seen when perfusing with FaSSIFHPMC, compared to FaSSIF. For tadalafil, a higher DS was achieved when perfusing with FaSSIFHPMC compared to FaSSIF. Perfusing the drugs with FaSSIFHPMC resulted in a significantly lower area under the curve (AUC0-60 min) for plasma concentrations of indomethacin, and no increase in the AUC0-60 min of plasma concentrations of tadalafil compared to perfusion with FaSSIF. The importance of simultaneously estimating the intraluminal DS and absorption of a drug was demonstrated by the SPIP model in the present study. Further, the study highlights the discrepancy between optimal in vitro supersaturation, intraluminal supersaturation and in vivo performance of two poorly soluble drugs, and further emphasizes the importance of optimization of in vitro methods in order to predict in vivo supersaturation and precipitation of drugs.

Using in vitro lipolysis and SPECT/CT in vivo imaging to understand oral absorption of fenofibrate from lipid-based drug delivery systems.

Using lipid-based drug delivery systems (LbDDS) is an efficient strategy to enhance the low oral bioavailability of poorly water-soluble drugs. Here the oral absorption of fenofibrate (FF) from LbDDS in rats was investigated in pharmacokinetic, in vitro lipolysis, and SPECT/CT in vivo imaging studies. The investigated formulations were soybean oil solution (SBO), a mixture of soybean oil and monoacyl phosphatidylcholine (MAPC) (SBO-MAPC), self-nanoemulsifying drug delivery systems with and without MAPC (SNEDDS-MAPC and SNEDDS, respectively), and an aqueous suspension (SUSP) as a reference. Oral bioavailability of the LbDDS ranged from 27 to 35%. A two-step in vitro lipolysis model simulating rat gastro-intestinal digestion provided in vitro FF solubilisation data to understand oral absorption. During the in vitro lipolysis, most FF was undissolved for SUSP and distributed into the poorly dispersed oil phase for SBO. For the SNEDDS without MAPC, practically all FF solubilised into the aqueous phase during the dispersion and digestion. Adding MAPC to SBO enhanced the dispersion of the oil phase into the digestion media while adding MAPC to SNEDDS resulted in a distribution of 29% of FF into the oil phase at the beginning of in vitro lipolysis. FF distribution into both oil and aqueous phases explained the higher and prolonged oral absorption of LbDDS containing MAPC. To elucidate the relatively low bioavailability of all formulations, FF and triolein were labeled with 123I and 125I, respectively, to study the biodistribution of drug and lipid excipients in a dual isotope SPECT/CT in vivo imaging study. The concentration of radiolabeled drug as a function of time in the heart correlated to the plasma curves. A significant amount of radiolabeled drug and lipids (i.e., 28-59% and 24-60% of radiolabeled drug and lipids, respectively) was observed in the stomach at 24 h post administration, which can be linked to the low bioavailability of the formulations. The current study for the first time combined in vitro lipolysis and dual isotope in vivo imaging to find the root cause of different fenofibrate absorption profiles from LbDDS and an aqueous suspension.

Developing a predictive in vitro dissolution model based on gastrointestinal fluid characterisation in rats.

The influence of physiological factors on the solubility of drug compounds has been thoroughly investigated in humans. However, as these factors vary between species and since many in vivo studies are carried out in rats or mice, it has been difficult to establish sufficient in vitro in vivo relations. The aim of this study was to develop a physiologically relevant in vitro dissolution model simulating the gastrointestinal (GI) fluids of fasted rats and compare it to previously published in vitro and in vivo data. To develop the in vitro model, the pH was measured in situ in six segments of the GI tract of anesthetised rats, then the fluids from the stomach, the proximal and the distal small intestine were collected and characterized with regard to osmolality, and bile acid and phospholipid concentration. The pH and osmolality were found to increase throughout the GI tract. The bile acids and phospholipids were present in high concentrations in the proximal small intestine, and the bile acid concentration doubled in the distal part, where the phospholipid concentration decreased. Matrix-assisted laser desorption ionisation mass spectrometry imaging was applied on a cross section of the small intestine, to study which bile acids and phospholipid classes were present in the small intestine of rats. Both cholic acid, taurocholic acid and glycocholic acid were detected, and phosphatidylcholine (34:2) was found to be mainly present in the intestinal wall or mucus, whereas lysophosphatidylcholine (16:0) was also detected in the lumen. Based on these observations, biorelevant media were developed to simulate fluids in the stomach and the proximal part of the small intestine in fasted rats. The media were implemented in a two-step in vitro dissolution model, which was found to better predict the in vivo performance of furosemide, when compared to previously published in vitro and in vivo data.

Characterization of the Hydrodynamics in a Miniaturized Dissolution Apparatus.

The hydrodynamics of a miniaturized dissolution apparatus was characterized using computational fluid dynamics simulations and analyzed in relation to the biorelevance and robustness of measurements of drug dissolution and precipitation kinetics from supersaturated drug solutions. The effect of using 3 different agitator geometries operated at 50, 100, 150, and 200 rpm as well as different positioning of an UV probe in the vessel was systematically evaluated. The computational fluid dynamics simulations were validated using a particle streak velocimetry experiment. The results show that the choice of agitator geometry influences the hydrodynamics of the system and indicates that an off-center probe position may result in more robust measurements. Furthermore, the study shows that the agitator geometry has a significant effect on supersaturation studies due to differences in the hydrodynamic shear produced by the agitator.

Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0µM. ß-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was ß-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of ß-alanine appear to have higher affinities for TauT than GABA-analogues.

Studying the Propensity of Compounds to Supersaturate: A Practical and Broadly Applicable Approach.

Supersaturating drug delivery systems can enhance the oral bioavailability of poorly soluble drug compounds. Supersaturation of such compounds has been studied in many different ways; however, a more standardized method is required. The rationale of choosing suitable concentrations of supersaturation to study has previously been very inconsistent. This makes comparisons between studies and compounds difficult, as the propensity of compounds to supersaturate varies greatly. This study presents a standardized method to study the supersaturation of drug compounds. The method allows, both, for a ranking of compounds according to their supersaturation propensity and the effectiveness of precipitation inhibitors. The time-concentration profile of supersaturation and precipitation was studied in situ for 4 different concentrations for 6 model compounds (albendazole, aprepitant, danazol, felodipine, fenofibrate, and tadalafil) in the µDISS Profiler™ in fasted-state simulated intestinal fluid. A relation between the induction time of nucleation and the initial supersaturated concentration could be established based on classical nucleation theory. The model compounds had different propensities to upersaturate. The data show that a single degree of supersaturation or concentration would not have described the different systems adequately. The method could be used in early preformulation for characterization of supersaturation propensity of novel compounds or precipitation inhibitor effects.

The use of supersaturation for the vaginal application of microbicides: a case study with dapivirine.

In this study, we investigated the potential of supersaturation for the formulation of the poorly water-soluble microbicide dapivirine (DPV) in an aqueous vaginal gel in order to enhance its vaginal tissue uptake. Different excipients such as hydroxypropylmethylcellulose, polyethylene glycol 1000, and cyclodextrins were evaluated for their ability to inhibit precipitation of supersaturated DPV in the formulation vehicle as such as well as in biorelevant media. In vitro permeation assessment across HEC-1A cell layers demonstrated an enhanced DPV flux from supersaturated gels compared with suspension gels. The best performing supersaturated gel containing 500 µM DPV (supersaturation degree of 4) in the presence of sulfobutyl ether-beta-cyclodextrin (2.5%) appeared to be stable for at least 3 months. In addition, the gel generated a significant increase in vaginal drug uptake in rabbits as compared with suspension gels. We conclude that supersaturation is a possible strategy to enhance the vaginal concentration of hydrophobic microbicides, thereby increasing permeation into the vaginal submucosa.

The anti-epileptic drug substance vigabatrin inhibits taurine transport in intestinal and renal cell culture models.

The GABA-mimetic anti-epileptic drug substance vigabatrin is used against infantile spasms. In vitro and in vivo experiments have shown that vigabatrin is transported via the proton coupled amino acid transporter (PAT1) mediating at least parts of the intestinal absorption of the drug. However, such evidence does not preclude the involvement of other transporters. The aim of the present study was, therefore, to investigate if vigabatrin interacts with taurine transport. The uptake of taurine was measured in intestinal human Caco-2 and canine MDCK cell monolayers in the absence or presence of amino acids such as GABA and vigabatrin. Vigabatrin inhibits the uptake of taurine in Caco-2 and MDCK cells to 34 ± 3 and 53 ± 2%, respectively, at a concentration of 30 mM. In Caco-2 cells the uptake of vigabatrin under neutral pH conditions is concentration-dependent and saturable with a Km-value of 27 mM (log Km is 1.43 ± 0.09). In conclusion, the present study shows that vigabatrin was able to inhibit the uptake of taurine in intestinal and renal cell culture models. Furthermore, uptake of vigabatrin in Caco-2 cells under neutral pH conditions was concentration-dependent and saturable and suggesting that vigabatrin partly was transported via a taurine transporter, which is likely to be TauT.